The clinically important anti-tumor agent 5-fluorouracil (5-FU) was designed to inhibit thymidylate synthase (TS). The potent inhibition of TS is an important aspect of the efficacy of 5-FU, but there is longstanding and growing evidence that 5-FU interferes with ribonucleic acid (RNA) mediated processes, particularly the splicing of precursor messenger RNA (pre-mRNA). It is possible that RNA directed actions of 5-FU constitute a major portion of its potency. The excision of intervening sequences and splicing of exons takes place in the spliceosome - a complex of small nuclear RNAs (snRNA) and associated proteins. Duplexes composed of single stranded regions of two or more different types of snRNA occur upon formation of the spliceosome. This association between snRNAs is based upon base pairing of complementary regions and is required for proper splicing of the pre-mRNA. These duplexes contain guanosme-uridine (GU) and uridine-uridine (UU) wobble base pairs in addition to guanosine- cytidine (GC) and adenosine-uridine (AU) base pairs. Fluorine substitution at C5 of U may impact the relative stability of wobble and normal base pairs and alter the binding stability of snRNA complexes containing 5-FU. The structural consequences of 5-FU incorporation into RNA are not well understood at present and the proposed analysis of RNA containing 5-FU by nuclear magnetic resonance (NMR) spectroscopy will contribute to our knowledge of the RNA directed mechanisms of potency and toxicity of this drug. This proposal aims to incorporate 5-fluorouridine into RNA oligomers using solution and solid-phase chemistries. A detailed model of the U4-U6 snRNA complex is developed based upon NMR spectroscopic data. The two stem regions formed by the interaction of human U4 and U6 snRNAs are studied in detail by using 1H NMR spectroscopy. The impact of 5-fluorouridine substitution for each uridine is assessed thermodynamically. Duplexes with anomalous melting profiles are studied in detail by using 1H NMR spectroscopy. A portion of the U4-U6 snRNA complex is prepared by using an in vitro T7 RNA polymerase transcription system. Isotopically enriched nucleoside triphosphate (NTP) is incorporated into the U4-U6 snRNA complex, which is then analyzed by 1H-13C and 1H-15N NMR spectroscopy. The overall impact of 5-FU incorporation on the U4-U6 snRNA complex is evaluated spectroscopically. Understanding the structural basis for the RNA mediated potency and toxicity of 5-FU will ald in the development of future anticancer strategies that rationally intervene in pre-mRNA splicing, or other RNA mediated cellular processes.